The ability of viruses to evade or antagonize the host type I IFN response plays an important role in viral tropism and disease pathogenesis. However, the precise cellular and molecular mechanisms by which viruses impede tissue specific host defenses leading to virus-induced pathology remain elusive. The coronavirus, mouse hepatitis virus (MHV), causes acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV protein ns2 blocks the IFN-inducible and potent antiviral 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L pathway in macrophages and facilitates the development of hepatitis. The ns2 protein and related viral (rotavirus VP3) and mammalian (AKAP7) proteins are 2H phosphoesterases with 2',5'-phosphodiesterase (PDE) activities that degrade 2-5A, the activator of RNase L. Our long-term objective is to probe the role of viral and host 2',5'-PDEs in antiviral innate immunity and its effect on viral pathogenesis. Our overall hypothesis herein is that the OAS-RNase L pathway in macrophages is critical in protecting the host from developing viral hepatitis. Our Specific Aims are: (1) To determine if RNase L signaling in liver macrophages (Kupffer cells, KC) restricts virus from entering the liver parenchyma and inducing hepatitis, we will compare MHV replication and activation of the OAS-RNase L pathway in primary cultures of KC, liver endothelial cells and hepatocytes, generate mice with cell type specific deletions of the RNase L gene and assess the development of hepatitis using wild type and ns2 mutant MHV. (2) To determine the roles of ns2 related viral and host 2',5'-PDEs, we will study their effects on 2-5A levels, viral replication, apoptosis and IFN- induction; and identify small molecule inhibitors of viral 2',5'-PDEs (ns2 and VP3) and cellular 2',5'-PDE (AKAP 7) by high throughput screening. (3) To determine and compare the proviral and RNase L antagonist activities of cellular and viral 2',5'-PDEs, we will investigate the mechanisms underlying 2',5'-PDE mediated protection to MHV by determining subcellular location of MHV replication complexes and ns2 and cellular AKAP7; generate chimeric viruses expressing different viral and cellular 2',5'-PDEs and determine their ability to antagonize RNase L and induce hepatitis. In addition, to determine effects of controlling 2-5A levels on viral pathogenesis during ongoing infections, virus-infected mice will be treated with inhibitors of 2',5'-PDEs. Our proposed studies will investigate a novel mode of regulation of the IFN antiviral response with broad implications for the control of viral infections that extend beyond hepatitis to virus mediated pathologies in other organ systems. Expected results include defining the role of KC as gatekeeper that prevents viruses from entering into the liver parenchyma and defining proviral and RNase L antagonist roles of diverse viral and cellular 2',5'-PDEs. It is anticipated that further understanding of the competition between virus and type I IFN signaling, particularly the OAS-RNase L pathway, will aid in the development or refinement of therapeutic strategies for human viral hepatitis and other viral diseases.